Pdgfαα

Mixed glial cells were generated from male and female P2-7 C57BL/6 pups according to the protocol of the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec). In short, brains were dissected, cerebellum and meninges were removed and tissue was dissociated via mechanical and papain enzyme digestion prior to filtration through a 40 μm strainer. Cells were plated in poly-L-lysine-coated (10 μg/ml, Sigma) 96 well flat, glass-bottomed plates (BD Falcon) at a density of 10⁵ cells per well and cultures were maintained at 37°C, 5% CO₂. Cells were cultured for 5 days in DMEM (Life Technologies) supplemented with PDGFαα (10 ng/ml; PeproTech), 10% endotoxin-free FCS, 1% penicillin/streptomycin and 1% L-glutamine (Life Technologies). Cells were cultured for a further 2 days in neural medium (Miltenyi Biotec) supplemented with 1% penicillin/streptomycin, 1% L-glutamine, 2% B27/MACS Neuro Brew21 (Miltenyi Biotec) and PDGFαα (10 ng/ml; PeproTech). At day 7 in culture, PDGFαα was withdrawn to allow oligodendrocyte differentiation and cells were stimulated with 5% T_reg-conditioned media, rCCN3, anti-CCN3 (clone 231216) or isotype control (clone 54447) (all R&D Systems) or controls for up to 5 days.

All the endogenous metabolites used in this study were from Sigma-Aldrich. Benztropine, miconazole and triiodothyronine (T3) were purchased from Sigma-Aldrich (Saint Louis, MO). All the chemicals used in this study have a purity ≥ 95%.
Rat primary optic nerve OPCs were isolated by panning (>99% A2B5⁺) and cultured in poly-d-lysine (10 µg/ml)-coated TC dishes in OPC culture media (Neurobasal Media, Invitrogen) supplemented with B27 without vitamin A (Invitrogen), 1× nonessential amino acids, 1× Glutamax, 1× anti-anti, β-mercaptoethanol and PDGFαα (50 ng/ml; Peprotech) at 37 °C with 5% CO₂. The culture medium was replaced every 48 h, and cells were collected before the confluency reached 60% to maintain a naive state. For differentiation, OPCs were plated at 1 million cells in one 10-cm Petri dish filled with differentiation media, which is identical to the culture media but with PDGF-AA at 2 ng/ml. T3 and DMSO were used as the positive control and negative control, respectively. Five replicates of cells were collected at different times (0, 3 and 6 d) for metabolomics studies.